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The Journal of Immunology, 2008, 180, 4939-4947
Copyright © 2008 by The American Association of Immunologists, Inc.
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IL-13 Attenuates Gastrointestinal Candidiasis in Normal and Immunodeficient RAG-2–/– Mice via Peroxisome Proliferator-Activated Receptor-{gamma} Activation1

Agnès Coste2,*,{dagger}, Céline Lagane2,*, Cédric Filipe§, Hélène Authier*, Amandine Galès*, José Bernad*, Victorine Douin-Echinard§, Jean-Claude Lepert*, Patricia Balard*, Marie-Denise Linas*,{ddagger}, Jean-François Arnal§, Johan Auwerx{dagger} and Bernard Pipy3,*

* Laboratoire Polarisation des Macrophages and Récepteurs Nucléaires dans les Pathologies Inflammatoires et Infectieuses, Université Paul Sabatier Toulouse III, Institut National de la Santé et de la Recherche Médicale (INSERM) Institut Fédératif de Recherche (IFR) 31, Institut Louis Bugnard, Toulouse, France; {dagger} Institut de Génétique et Biologie Moléculaire et Cellulaire, INSERM/Centre National de la Recherche Scientifique, Illkirch, France; {ddagger} Département de Parasitologie et Mycologie, Centre Hospitalier Universitaire, Hôpital Rangueil, Toulouse, France; and § INSERM Unité 858, IFR31, Institut Louis Bugnard, Toulouse, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We recently demonstrated that in vitro peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) activation of mouse peritoneal macrophages by IL-13 or PPAR{gamma} ligands promotes uptake and killing of Candida albicans through mannose receptor overexpression. In this study, we demonstrate that i.p. treatment of immunocompetent and immunodeficient (RAG-2–/–) mice with natural and synthetic PPAR{gamma}-specific ligands or with IL-13 decreases C. albicans colonization of the gastrointestinal (GI) tract 8 days following oral infection with the yeast. We also showed that Candida GI infection triggers macrophage recruitment in cecum mucosa. These mucosal macrophages, as well as peritoneal macrophages, overexpress the mannose receptor after IL-13 and rosiglitazone treatments. The treatments promote macrophage activation against C. albicans as suggested by the increased ability of peritoneal macrophages to phagocyte C. albicans and to produce reactive oxygen intermediates after yeast challenge. These effects on C. albicans GI infection and on macrophage activation are suppressed by treatment of mice with GW9662, a selective PPAR{gamma} antagonist, and are reduced in PPAR{gamma}+/– mice. Overall, these data demonstrate that IL-13 or PPAR{gamma} ligands attenuate C. albicans infection of the GI tract through PPAR{gamma} activation and hence suggest that PPAR{gamma} ligands may be of therapeutic value in esophageal and GI candidiasis in immunocompromised patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Candida albicans is part of the microbial flora that colonizes the mucocutaneous surfaces of the oral cavity and gastrointestinal (GI)4 tract of many mammals as well as other organisms (1). This yeast represents a serious issue in immunocompromised patients and those undergoing immunosuppressive therapies, particularly with impaired phagocytic cell function (mainly neutrophils, monocytes, and macrophages). The innate immune system initiated by macrophages is therefore essential for orchestrating events associated with prevention of fungal colonization. Macrophages play a direct role in microbial control through their ability to phagocytose yeasts and to exert their fungicidal activity by releasing large amounts of highly toxic molecules, such as reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates. These two mechanisms involve the macrophage mannose receptor (MR), a key pattern-recognition receptor of innate immunity (2). Indeed, this receptor is both a major phagocytic and an endocytic receptor sufficient to mediate binding and phagocytosis of a range of unopsonized microorganisms bearing terminal mannose, fucose, or N-acetylglucosamine residues on the cell surface of potential pathogens such as C. albicans (3), Pneumocystis carinii (4), Klebsiella pneumoniae (5). Binding or internalization of natural or synthetic ligands of MR may modulate macrophage functions, including respiratory burst (6) and synthesis of proinflammatory cytokines, such as IL-1β, IL-6, and GM-CSF (7) to increase their microbicidal capacity. Previous studies have shown that MR expression can be positively modulated in vitro by many agents, in particular by 1,25-dihydroxyvitamin D3 (8), prostaglandin E2 (9), IL-4, and IL-13 (10, 11). Further support for a role of IL-13 in the control of the innate immune response came from the characterization of susceptibility to infection of animals that were deficient in IL-13. IL-13–/– mice failed to clear GI nematode infections efficiently (12), suggesting that IL-13 could also be an important endogenous mediator of microbial resistance. The classical IL-13-signaling pathway involves transcription factor STAT-6. This was first shown for 12/15-lipoxygenase, CD23, CD13, CD11b, and class II MHC up-regulation (13, 14). But other transcription factors can be involved in IL-13 signaling. We previously reported that the increase of macrophage MR expression by IL-13 involved peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) activation via a PLA2 signaling pathway (6). In the same study, an unanticipated antimicrobial action of PPAR{gamma} resulted from of cross-talk between PPAR{gamma} and the in vitro regulation of macrophage MR expression through which PPAR{gamma} contributes to increased recognition and phagocytosis of unopsonized C. albicans. Although the role of PPAR{gamma} in the regulation of adipogenesis (15), lipid homeostasis, and monocyte gene expression and differentiation (16) is relatively well-established, its involvement in antimicrobial defense has not been explored.

In this study, we validate the potent in vivo immunostimulatory role of natural and synthetic PPAR{gamma}-specific ligands and IL-13 to fight C. albicans colonization in experimental GI candidiasis. We demonstrate that in vivo treatment with PPAR{gamma} ligands and IL-13 significantly decreases Candida infection in the GI tract both in immunocompetent and immunodeficient RAG-2–/– mice. The use of RAG-2–/– mice, lacking T and B cells, demonstrates the predominant role of macrophages in these mechanisms. Moreover, the reduction of C. albicans colonization of the digestive tract by PPAR{gamma} ligands and IL-13 is correlated with an increase of MR expression on recruited mucosal macrophages. These data demonstrate for the first time that the in vivo regulation of macrophage MR by IL-13 and PPAR{gamma} ligands is linked to the transcription factor PPAR{gamma} and strongly suggests that macrophage MR is responsible for C. albicans elimination by improving microbicidal functions of macrophages, such as phagocytosis and ROI synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Microorganism

The strain of C. albicans used throughout these experiments was isolated from the blood culture of a patient in Toulouse-Rangueil Hospital. The isolate was identified as C. albicans based on common laboratory criteria and cultured on Sabouraud dextrose agar (SDA) plates containing gentamicin and chloramphenicol. The strain was maintained by transfers on SDA plates. Growth from an 18- to 24-h SDA culture of C. albicans was suspended in sterile saline at a concentration of 1 x 108 cells/ml, as determined by hemacytometer counts and verified by plate counts.

Mice and infection procedure

All animal experimentation was conducted in accordance with accepted standards of humane animal care. Mouse experiments were approved and performed according to the guidelines of the Toulouse University Animal Safety Committee and of the Regional Safety Committee (Procedure 1992). The model of GI candidiasis was established on 6-wk-old female wild-type C57BL/6 mice and immunodeficient RAG-2–/– C57BL/6 mice (Centre National de la Recherche Scientifique, Orléans, France) or wild-type SV129 mice and PPAR{gamma}+/– SV129 mice (Institut Clinique de la Souris, Illkirch, France). Because the RAG-2–/– animals have no B or T cell-mediated immunity (17), they were housed in sterile microisolator cages, with autoclaved food, sterile water ad libitum, bedding and under defined flora that is nonpathogenic. All cages were changed twice weekly, and all manipulations of the animals were done in a laminal blow hood under aseptic conditions. The photoperiod was adjusted to 12 h light and 12 h dark. To establish esophageal and GI candidiasis in mice, we performed intraesophageal infection with 5 x 107 viable cells of C. albicans in sterile saline solution (500 µl/mouse). No antibiotic or immunosuppressive treatment was used to facilitate mucosal infection by C. albicans of the oral cavity and GI tract.

Treatment groups

Therapeutic studies were performed on separate groups of six mice, each infected, or not, by C. albicans. Wild-type, RAG-2–/–, and PPAR{gamma}+/– mice received the following products in 500 µl of 0.9% NaCl by i.p. route. Final DMSO concentration was lower than 0.1% (v/v). Mice were treated with IL-13 (Sanofi) 1 day before infection with C. albicans at a final concentration of 5 µg/mouse. For treatment with PPAR{gamma} ligands, 15d-PGJ2 (Cayman Chemical) and rosiglitazone (Cayman Chemical), mice were injected 1 day before infection, the day of infection, and then every 2 days at a final concentration of 0.1 µM per mouse. GW9662 (Cayman Chemical), a PPAR{gamma} antagonist, was administered 30 min before IL-13 or rosiglitazone injections at a final concentration of 220 µM/mouse. Control groups received saline solution only with DMSO. The body weight of each mouse was recorded daily, and the condition of each mouse was assessed twice daily. On day 8 after treatments (7 days after infection), all mice were euthanized using CO2 asphyxia. At this time point, esophagus, stomach, cecum, liver, and kidneys were aseptically removed to evaluate C. albicans colonization.

Quantification of the number of viable C. albicans in the esophagus, GI tract, and visceral organs

Each tissue sample removed was mechanically homogenized in 1 ml of saline with 100 U of penicillin/ml and 100 µg of streptomycin/ml. Serial dilutions of homogenate were plated onto SDA for quantitative determination of the number of C. albicans in the tissue samples. Plates were incubated at 37°C for 1–2 days and the number of colonies was counted. The number of CFU in each organ was determined.

Quantification of C. albicans in the esophagus, GI tract, and visceral organs using a quantitative real-time PCR

Cell lysis and DNA extraction. A total of 100 µl of each tissue sample homogenate was resuspended in 800 µl of lysis buffer (0.05 M EDTA, 0.1 M SDS, 0.05 M Tris-HCl, 1% 2-ME) for 3 h at 65°C (18). DNA was extracted with phenol-chloroform isoamyl alcohol, then 4.5 ml of 5 M ammonium acetate followed by the addition of 2 volumes of 100% ethanol. DNA was precipitated for 1 h at –80°C and recovered by centrifugation for 20 min at 12,000 x g. A gentle 70% ethanol wash was performed, and the pellets were then dried under vacuum for 10 min. These DNA pellets were taken up in 100 µl of sterile deionized water and allowed to resuspend overnight. After DNA extraction, 3 µl of samples of the same mouse groups were pooled to perform the quantification by PCR.

Light Cycler-based PCR assay. The Light Cycler PCR and detection system (Roche Diagnostics) was used for amplification and online quantification. PCR was performed in glass capillaries, ensuring rapid equilibration between the air and the reaction components because of the high surface-to-volume ratio of the capillaries. Primers (5'-ATT GGA GGG CAA GTC TGG TG and 5'-CCG ATC CCT AGT CGG CAT AG; Eurogentec) bind to conserved regions of the fungal 18S rRNA gene as described previously (19). For amplicon detection, the Light Cycler DNA Master Hybridization Probes kit was used as described by the manufacturer. Briefly, two different oligonucleotides hybridize to an internal species-specific sequence of the 18S rRNA gene of C. albicans. One probe was labeled at the 5' end with the Light Cycler Red 640 fluorophore (5'-TGG CGA ACC AGG ACT TTT ACT TTG A) (Tibmolbiol), and the other was labeled at the 3' end with fluorescein (5'-AGC CTT TCC TTC TGG GTA GCC ATT; Tibmolbiol) (20). During fluorescence resonance energy transfer fluorescein is excited by the light source of the Light Cycler instrument. The excitation energy is transferred to the acceptor fluorophore, Light Cycler Red 640, and the emitted fluorescence is measured after annealing by the photohybrids of the instrument. The PCR mixture contained Taq polymerase, 1x Light Cycler hybridization reaction buffer, a deoxynucleoside triphosphate mixture (with dUTP instead of dTTP), 3 mM magnesium chloride, and 12.5 pM of primers. Amplification was performed for 45 cycles of repeated denaturation (1 s at 95°C), annealing (15 s at 62°C), and enzymatic chain extension (25 s at 72°C).

Light Cycler-based quantification of target DNA. Quantification was performed by online monitoring for identification of the exact time point at which the logarithmic linear phase could be distinguished from the background (crossing point). Serially diluted samples of genomic fungal DNA obtained from C. albicans cultures (106–100 CFU) were used as external standards in each run. Cycle numbers of the logarithmic linear phase were plotted against the logarithm of the concentration of template DNA to evaluate the number of yeast cells present in each tissue sample homogenate.

Preparation of mouse resident peritoneal macrophages

After euthanasia, resident peritoneal cells were harvested by washing the peritoneal cavity with 5 ml of sterile 199 medium with Hanks’ salts as previously described (6). Collected cells were centrifuged at 400 x g for 8 min and the cell pellet was suspended in serum-free medium optimized for macrophage culture (Invitrogen Life Technologies). In all our experiments, before measuring phagocytosis and ROI production, the macrophages were counted in a hemacytometer and their numbers made even in each well. Cells were allowed to adhere onto 24- or 96-well culture plates for 1 h at 37°C and 5% CO2. Nonadherent cells were removed by washing with PBS (Invitrogen Life Technologies Corporation, France). After 2 h of culture, >98% of the adherent cells thus obtained had the appearance of macrophages after May-Grunwald Giemsa staining and were detected as positive for nonspecific esterase. The macrophage monolayers were used to investigate phagocytosis capacity and ROI production after Candida albicans challenge in vitro and to evaluate MR surface expression.

Phagocytosis assay

To investigate the mechanism of C. albicans ingestion by murine peritoneal macrophages, we used [3H]uracil-prelabeled yeasts. Three C. albicans colonies were dispersed in 1 ml of Sabouraud broth containing 150 µCi [3H]uracil. After 24 h of growth at 37°C, the pellet was resuspended in serum-free medium supplemented with glucose 1.8%. Macrophages collected from mice untreated or treated in vivo by IL-13, 15d-PGJ2, rosiglitazone, and/or GW9662 were plated in 24-well Falcon plates (5 x 105 macrophages/well). They were then infected with viable, labeled C. albicans for 1 h (ratio of macrophage: yeast, 1:1) at 37°C to study Candida internalization by the macrophages and at 5°C to study Candida-cell adhesion. After an incubation period, culture medium was collected, and monolayers were washed twice with PBS. The monolayers were disrupted as previously described (6). The radioactivity of the supernatant and washings, and the radioactivity contained in the cellular lysate, were counted independently. An index of phagocytosis was calculated with the following equation: percent phagocytosis = {Delta}(37°C–5°C) dpm monolayers/({Delta}(37°C–5°C) dpm monolayers + dpm supernatant).

Assay for oxidizing agent production

The macrophages from untreated or IL-13-, 15d-PGJ2-, rosiglitazone-, and/or GW9662-treated mice were plated in 96-well Falcon plates (1.5 x 105 macrophages/well). Oxygen-dependent respiratory burst of macrophages collected from mice untreated or treated in vivo by IL-13, 15d-PGJ2, rosiglitazone, and/or GW9662 was measured by chemiluminescence (CL) in the presence of 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol) using a thermostatically (37°C) controlled luminometer (Wallac 1420 Victor2). CL generation was monitored continuously for 1 h after incubation of 1.5 x 105 macrophages with luminol (66 µM) and after C. albicans challenge (1:1). Statistical analysis was performed using the area under the curve expressed in counts x second.

MR surface expression

To evaluate MR surface expression on mouse peritoneal macrophages harvested from mice untreated or treated in vivo by IL-13, 15d-PGJ2, rosiglitazone, and/or GW9662, 1 x 106 peritoneal cells were incubated directly with a mannosylated BSA (mBSA), an MR-specific ligand, conjugated with FITC (840 nM) (7, 11). Labeling of MR was done during 1 h on ice. We checked that binding of the FITC-mannose-BSA could be fully reversed by mannose-BSA without FITC or yeast mannan addition but not by BSA. To facilitate the selection of a viable cell population, staining was done with propidium iodide. All analyses were done on a BD Biosciences FACScan using CellQuest software. A population of 20,000 viable cells was analyzed for each data point.

Immunohistochemical analysis of cecum macrophages

Cecum tissues of wild-type mice infected or not with C albicans and treated or not with rosiglitazone or IL-13 were removed, and carefully opened longitudinally. After washing in PBS, the tissues were fixed for 20 min in PBS containing 4% paraformaldehyde. Then, fixation was quenched with 100 mM/L glycine (pH 7.4), and samples were permeabilized for 10 min in Triton X-100 and washed in PBS. Fixed tissues were blocked with solution I (75 mM NaCl, 18 mM Na3 citrate, 2% goat serum, 1% BSA, 0.05% Triton X-100, and 0.02% NaN3) for 2 h at room temperature. The tissues were first stained with CD68 by incubating cecum samples with CD68 Ab rat anti-mouse; Serotec) diluted (1/100) in solution I for 48 h at 4°C. Then, tissues were washed in solution II (75 mM NaCl, 18 mM sodium citrate, and 0.05% Triton X-100) for 2 h and rinsed in PBS. The tissues were then incubated for 1 h at room temperature with Alexa 633-conjugated goat anti-rat Ab (5 µg/ml; Molecular Probes) diluted in solution I. Finally, they were washed in solution I for 1 h at room temperature. The same protocol was repeated for anti CD206 labeling (rabbit anti-mouse, 1:250; Santa Cruz Biotechnology), and the second Ab (goat anti-rabbit; Sigma-Aldrich). To label the nuclei, propidium iodide was used (2 µg/ml; Sigma-Aldrich). All preparations were mounted with Kaiser’s glycerol gelatin (Merck). All microscopy imagery was performed with a Zeiss LM 510 (Carl Zeiss).

Statistical analysis

Data are expressed as mean ± SE of six different animals. For each experiment, data were subjected to one-way ANOVA followed by the means multiple comparison method of Tukey. A value of p < 0.05 was considered as the level of statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
In vivo treatments of normal and immunodeficient RAG-2–/– mice with PPAR{gamma} ligands and IL-13 decreased C. albicans colonization of the GI tract

To characterize the in vivo effectiveness of 15d-PGJ2, rosiglitazone, and IL-13 in treating fungal infection, we subjected immunocompetent and immunodeficient mice to oral inoculation with C. albicans. In this study, to model infections that closely mimic diseases found in human HIV+ patients, we used a mouse strain with MLR2 knockout (RAG-2–/–). As in AIDS patients, B and T cell-mediated immune mechanisms are absent and only the cells of innate immunity, such as macrophages, remain effective. In this model of candidiasis, both in immunocompetent and in immunodeficient mice, yeasts extensively colonized the esophagus and the GI tract. No yeasts were recovered in homogenates of other internal organs (kidney and liver), demonstrating the absence of disseminated disease.

As expected, the number of yeasts recovered in esophagus and cecum was significantly increased in immunodeficient RAG-2–/– mice as compared with immunocompetent mice (Fig. 1, A–C), demonstrating that B and T cell-mediated immune mechanisms are important in limiting proliferation of C. albicans in these organs. Interestingly, after treatment with 15d-PGJ2, rosiglitazone, or IL-13, the number of yeasts present in esophagus, stomach, and cecum was significantly decreased both in immunocompetent and immunodeficient RAG-2–/– mice (Fig. 1, A–C). Similar conclusions were obtained from C. albicans DNA quantification by quantitative RT-PCR, which demonstrated that the various treatments decreased C. albicans colonization of esophagus and GI tract from immunocompetent and immunodeficient RAG-2–/– mice (Fig. 1, D–F). This clearly demonstrates the protective effect of 15d-PGJ2, rosiglitazone, and IL-13 treatments against C. albicans mucosal colonization.


Figure 1
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  		FIGURE 1. Intraperitoneal treatment of immunocompetent or RAG-2–/– immunocompromised mice with PPAR{gamma} ligands or IL-13 decreased esophagus and GI tract C. albicans colonization. C. albicans colonization of esophagus (A and D), stomach (B and E), and cecum (C and F) from immunocompetent (WT) and immunocompromised (RAG-2–/–) untreated or 15d-PGJ2-, rosiglitazone- (Rosi), or IL-13-treated mice evaluated by CFUs (A–C) and by quantitative RT-PCR (D–F) (n=6). **, p < 0.01, a significant difference compared with the respective control groups (WT and RAG-2–/– untreated mice). #, p < 0.05, and ##, p < 0.01, a significant difference compared, respectively, with WT untreated or 15d-PGJ2-, rosiglitazone-, and IL-13-treated mice. For quantitative RT-PCR, DNA samples extracted from each mouse of the same group were pooled. The quantity of yeast DNA was determined using Light-Cycler technology. The yeast number of each tissue sample homogenate was calculated with a standard curve using various concentrations of yeasts analyzed in parallel in the same run as indicated in Materials and Methods.

 
IL-13 and rosiglitazone treatments trigger recruitment to cecal mucosa of macrophages that overexpress MR

Because MR is the main pattern-recognition receptor involved in mannan recognition and its overexpression by IL-13 or rosiglitazone leads in vitro to C. albicans elimination (6), we evaluated the recruitment of macrophages expressing MR to the digestive mucosa of mice infected with C. albicans and treated with IL-13 or rosiglitazone. Fig. 2, B and C, showed that in the absence of yeasts and treatment (Fig. 2B, upper left panel), CD68-positive cells (CD68+) were not present in the cecal mucosa. However, with IL-13 treatment (Fig. 2B, lower left panel), but not with rosiglitazone (Fig. 2B, middle left panel), the number of CD68+ cells increased (3.77 ± 0.31% of total cells), indicating that the IL-13 treatment recruited CD68+ cells (p < 0.01). After C. albicans inoculation, the presence of CD68+ cells increased significantly (Fig. 2B, right panels), attesting to the capacity of the yeasts to recruit CD68+ cells in the cecum mucosa. This experiment also demonstrated that after C. albicans inoculation, in the absence of treatment (Fig. 2B, upper right panel), these cells expressed the MR slightly (2.88 ± 0.13% of total cells), and that treatment with either rosiglitazone or IL-13 significantly increased the number of CD68+MR+ cells, respectively, by 6.04 ± 0.25% (Fig. 2B, middle right panel) and 9.48 ± 0.93% (Fig. 2B, lower right panel) (p < 0.01 according to control group, infected nontreated mice). These results indicated that IL-13 and rosiglitazone treatments favor the infiltration of macrophages in the cecal mucosa with a strong proportion of type 2 macrophages (M2).


Figure 2
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  		FIGURE 2. Intraperitoneal treatment of mice with PPAR{gamma} ligands or IL-13 favor the recruitment of macrophages expressing MR in the cecal mucosa. Confocal laser microscopy of cecum tissues from untreated, rosiglitazone-, IL-13-treated mice infected with C. albicans or not. A, The blue color represents the nucleus; red represents CD68-positive cells; green represents MR-positive cells. Merged picture of green and red colors represents CD68 and MR-positive cells. B, Confocal laser microscopy of the top of the cecum villosity from untreated (upper panels), rosiglitazone (middle panels), IL-13- (bottom panels) treated mice infected (right panels) or not (left panels) with C. albicans. Stainings are representative of at least three different cecal tissue samples of three animals by group. C, The percentage of CD68-positive cells or CD68 and MR-positive cells (n = 3). **, p < 0.01, a significant difference compared with the respective control groups (untreated mice noninfected or infected with C. albicans).

 
In vivo treatment of normal and immunodeficient RAG-2–/– mice with PPAR{gamma} ligands and IL-13 induces overexpression of macrophage MR and their associated effector functions

To precisely assess that PPAR{gamma} ligands (rosiglitazone and 15d-PGJ2) and IL-13 promote the expression of MR on macrophages in vivo, we analyzed by flow cytometry, the induction of MR expression on murine peritoneal macrophages from wild-type or RAG-2–/– mice treated with IL-13, 15d-PGJ2, or rosiglitazone. As shown in Fig. 3A, the amount of labeled mBSA-FITC on peritoneal macrophages collected from in vivo 15d-PGJ2- or rosiglitazone-treated normal or immunodeficient RAG-2–/– mice was significantly increased as compared with macrophages of untreated mice. Similarly, labeling was significantly higher following treatment with IL-13. The specificity of mBSA for MR suggests that in vivo treatment with PPAR{gamma} ligands or IL-13 increases the MR protein level on the surface of peritoneal macrophages of both immunocompetent and immunodeficient mice.


Figure 3
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  		FIGURE 3. Intraperitoneal treatment of immunocompetent or RAG-2–/– immunocompromised mice with PPAR{gamma} ligands or IL-13 increased macrophage MR expression, C. albicans phagocytosis, and oxidant production of peritoneal macrophages. A, Mannose receptor surface expression on peritoneal macrophages from immunocompetent (WT) or immunocompromised (RAG-2–/–) untreated or 15d-PGJ2-, rosiglitazone-, and IL-13- treated mice (n = 6). Cells were incubated with mannosylated BSA-FITC and analyzed by flow cytometry. B, Phagocytosis capacity of peritoneal macrophages from immunocompetent (WT) or immunocompromised (RAG-2–/–) untreated or 15d-PGJ2-, rosiglitazone-, and IL-13-treated mice (n = 6). The ability of peritoneal macrophages to phagocytose C. albicans was evaluated after 1 h of challenge with yeasts labeled with [3H]uracil. C, ROI production of peritoneal macrophages from immunocompetent (WT) or immunocompromised (RAG-2–/–) untreated or 15d-PGJ2-, rosiglitazone- (Rosi), and IL-13-treated mice (n=6). Total CL emission was evaluated continuously for 60 min in the absence or presence of C. albicans (n = 6). **, p < 0.01, a significant difference compared with the respective control groups (WT and RAG-2–/– untreated mice). #, p < 0.05, and ##, p < 0.01, a significant difference compared, respectively, with WT untreated or 15d-PGJ2-, rosiglitazone-, and IL-13-treated mice.

 
Because the mechanisms of defense against Candida are mainly dependent on the ability of macrophages to phagocytose yeasts and to exert their fungicidal activity by releasing large amounts of highly toxic molecules such as reactive oxygen intermediates, we studied the modulation of Candida phagocytosis and oxidant agent production of macrophages from untreated, 15d-PGJ2, rosiglitazone, or IL-13-treated mice. Fig. 3B shows that when macrophages were harvested on 15d-PGJ2- or rosiglitazone-treated immunocompetent or immunodeficient RAG-2–/– mice, the number of [3H]uracil-prelabeled C. albicans ingested increased significantly compared with macrophages from untreated mice. Similar results were obtained from macrophages of IL-13-treated immunocompetent or immunodeficient RAG-2–/– mice. Hence, these studies suggested that in vivo treatments of mice with PPAR{gamma} ligands and IL-13 significantly stimulate the capacity of macrophages to take up C. albicans. Regression analysis of the data described in Fig. 3, A and B, showed significant linear regressions (r2 = 0.987 for normal mice and r2 = 0.972 for immunodeficient RAG-2–/– mice) between MR fluorescence and the index of phagocytosis of macrophages, demonstrating a true correlation between macrophage MR expression influenced by different treatments and the ability of these cells to take up C. albicans.

Using CL, we next investigated the effect of in vivo 15d-PGJ2, rosiglitazone, and IL-13 treatment on macrophage oxidizing agent production. Fig. 3C shows that in vivo treatment of both immunocompetent and immunodeficient RAG-2–/– mice had no effect on the basal production of oxidizing agents by macrophages compared with macrophages from untreated mice. Addition of yeasts to macrophages from untreated mice did not increase the production of oxidizing agents, but significantly increased ROI production by macrophages from 15d-PGJ2-, rosiglitazone-, and IL-13-treated immunocompetent and immunodeficient RAG-2–/– mice. These data therefore suggest that these treatments preactivate the capacity of macrophages to produce oxidizing agents in response to C. albicans infection.

PPAR{gamma} antagonist inhibits macrophage MR overexpression and candidacidal activity induced in vivo by IL-13 and rosiglitazone treatments

To assess the mechanisms involved in the increase of MR expression and microbicidal functions of macrophages from IL-13- or PPAR{gamma} ligand-treated mice, we subjected mice to i.p. injections with GW9662, an irreversible antagonist of PPAR{gamma}. As expected, in vivo IL-13 and rosiglitazone treatment significantly increased MR expression on peritoneal macrophages (Fig. 4A). Interestingly, the increase was totally abolished by in vivo GW9662 pretreatment. This strongly suggests that in vivo PPAR{gamma} activation is involved in the up-regulation of macrophage MR expression mediated by IL-13 or rosiglitazone.


Figure 4
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  		FIGURE 4. Treatment of mice with a specific PPAR{gamma} antagonist, GW9662, suppresses the increase of macrophage MR expression, C. albicans phagocytosis, and oxidant production of macrophages induced by rosiglitazone and IL-13. A, Mannose receptor surface expression on peritoneal macrophages from untreated or rosiglitazone- (Rosi) and IL-13-treated mice, cotreated with GW9662 or not (n = 6). The cells were incubated with mBSA-FITC and analyzed by flow cytometry. B, Phagocytosis capacity of peritoneal macrophages from untreated or rosiglitazone- and IL-13-treated mice, cotreated with GW9662 or not (n = 6). The capacity of peritoneal macrophages to phagocytose C. albicans was evaluated after 1 h of challenge with yeasts labeled with [3H]uracil. C, ROI production of peritoneal macrophages from mice untreated or treated with rosiglitazone and IL-13, and cotreated with GW9662 or not (n = 6). Total CL emission was evaluated continuously for 60 min. * (p < 0.05), ** (p < 0.01) represents a significant difference compared with the control group (untreated mice). # (p < 0.05), ## (p < 0.01) represent a significant difference compared with rosiglitazone- or IL-13-treated mice without GW9662. C. albicans colonization of (D) stomach and (E) cecum from untreated or rosiglitazone- and IL-13-treated mice, cotreated or not with GW9662, evaluated by quantitative RT-PCR (n = 6). DNA samples extracted from each mouse of the same group were pooled. The quantity of yeast DNA was determined using Light-Cycler technology. The number of yeast cells in each tissue homogenate was calculated from a standard curve plotted using various concentrations of yeast analyzed in parallel in the same run, as indicated in Materials and Methods.

 
To further consolidate our hypothesis that PPAR{gamma} promotes the regulation of MR expression and hence activates the effector functions of macrophages, we studied the capacity of phagocytosis and ROI production of rosiglitazone- or IL-13-treated macrophages in response to C. albicans infection in the presence of GW9662. As predicted, the increase of both the phagocytosis index and the production of the oxidizing agents by macrophages treated by rosiglitazone or IL-13 were inhibited by GW9662 (Fig. 4, B and C). Consistent with our previous results, when animals received GW9662, the reduction of C. albicans in stomachs and cecums induced by IL-13 or rosiglitazone treatments is impaired, indicating a major contribution of PPAR{gamma} activation in the induction of fungicidal activity by in vivo IL-13 and rosiglitazone treatments (Fig. 4, D and E). These data therefore provide strong arguments for the in vivo involvement of PPAR{gamma} in the increase of MR expression and hence in the fungicidal activity of macrophages treated by rosiglitazone and IL-13.

Macrophage MR overexpression and C. albicans elimination are impaired in PPAR{gamma}+/– mice

The previous data were all consistent with an involvement of PPAR{gamma} in the induction of macrophage MR expression and in fungicidal activity of IL-13 and rosiglitazone, and incited us to evaluate whether these treatments were effective in a genetic model of mice. We used mice presenting a very low expression of PPAR{gamma} (PPAR{gamma}+/– mice), as determined by Western blot and quantitative RT-PCR (data not shown). IL-13 and rosiglitazone in vivo treatments significantly increased MR expression on peritoneal macrophages from wild-type mice (PPAR{gamma}+/+ mice) (Fig. 5A). However, this induction of MR expression, obvious in macrophages collected on rosiglitazone- and IL-13-treated PPAR{gamma}+/+ mice, was not observed on rosiglitazone- and IL-13-treated PPAR{gamma}+/– mice (Fig. 5A). In line with this result, the reduction of C. albicans colonization in stomachs and cecums after IL-13 or rosiglitazone treatments of PPAR{gamma}+/+ mice is impaired in PPAR{gamma}+/– mice (Fig. 5, B and C). These data provided strong arguments for the in vivo involvement of the PPAR{gamma} pathway in the increase of antimicrobial response induced by IL-13 or rosiglitazone treatment.


Figure 5
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  		FIGURE 5. The increase of macrophage MR expression and the decrease of GI C. albicans colonization by rosiglitazone and IL-13 were impaired in PPAR{gamma}+/– mice. A, Surface expression of MR on peritoneal macrophages from homozygous (PPAR{gamma}+/+) or hemizygous (PPAR{gamma}+/–) untreated or rosiglitazone- (Rosi) and IL-13-treated mice (n = 6). Cells were incubated with mBSA-FITC and analyzed by flow cytometry. **, (p < 0.01) represents a significant difference compared with the control group (untreated PPAR{gamma}+/+ mice). ## (p < 0.01) represents a significant difference compared with rosiglitazone- or IL-13-, PPAR{gamma}+/+-treated mice. C. albicans colonization of (B) stomach and (C) cecum from macrophages from homozygous (PPAR{gamma}+/+) or hemizygous (PPAR{gamma}+/–) untreated or rosiglitazone- and IL-13-treated mice (n = 6). DNA samples extracted from each mouse of the same group were pooled. The quantity of yeast DNA was determined by Light-Cycler technology. The number of yeast cells in each tissue homogenate was calculated with a standard curve plotted using various concentrations of yeast analyzed in parallel in the same run, as indicated in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Candida albicans is a fungus that can exist as a harmless colonizer or as an opportunistic pathogen, depending on the status of its host (21). So, C. albicans usually causes disease in immunocompromised hosts such as leukemic, organ-transplanted, diabetic, and HIV-infected patients. In immunocompetent hosts, innate and acquired humoral and cellular immune mechanisms are involved in resistance to candidiasis. Involvement of cell-mediated immunity in the control of infection is considered to be important for resistance to mucocutaneous and systemic candidiasis. In this process, macrophages serve as the effector cells of an immune response orchestrated by T cells (22). Exposure of macrophages to either Th1 or Th2 cytokines induces two distinct pathways of macrophage activation (23, 24). Classically, protective immunity to C. albicans infection results from the development of Th1 responses (25). So, cytokines produced by Th1 cells, such as IFN-{gamma}, activate phagocytic cells to a candidacidal state, increasing the secretion of effector molecules such as ROIs (10). Th2 cytokines, IL-4 and IL-13, were subsequently found to be more than simple inhibitors of macrophage activation, as they elicit a different activation program, termed macrophage alternative activation (23). Alternatively activated macrophages express MR, arginase I, FIZZ-1, and Ym-1 (23). The role of these macrophages alternatively activated by IL-13 in C. albicans killing has been recently described (6, 10). Indeed, we showed previously that in an unopsonized context, as found in immunocompromised hosts with no T or B cells, in vitro IL-13-treated macrophages express a high level of MR and hence attenuate C. albicans proliferation by increasing their ability to phagocytose the yeasts and to produce ROI but not NO (10). Although Th2 cytokines are clearly responsible for in vitro macrophage alternative activation, few works show the in vivo involvement of these macrophages in infectious processes.

In the present study, we used for the first time, the RAG-2–/– mouse model to evaluate the in vivo efficacy of IL-13 and PPAR{gamma} ligands in the treatment of mucosal candidiasis in the context of severe immunosuppression. RAG-2–/– animals are genetic knockouts for the RAG-2–/– gene and hence are unable to join V, D, and J segments to generate functional Ig or TCRs. Consequently, in these mice mature T and B cells are absent and the animals are unable to develop either specific Ab or cell-mediated responses. In contrast, cells involved in nonspecific immunity, such as neutrophils and monocytes, are intact and fully functional (26). Here, we demonstrated that RAG-2–/– mice are more susceptible to sustained Candida colonization of esophagus and cecum than immunocompetent mice although these mice express higher MR and have increased phagocytic activity, suggesting that the innate immune response does not compensate totally the lack of lymphocytes. Moreover, these mice did not develop disseminated infection from the sustained colonization and survived 30 days after oral infection. Thus, the model closely reproduces the mucosal candidiasis found in HIV/AIDS patients. Indeed, among these patients, esophagus and GI tract are strongly colonized while systemic dissemination rarely occurs (27, 28). Our results in RAG-2–/– mice are in line with other studies using SCID mice. These mice, which mimic the mucosal candidiasis observed clinically in AIDS patients, develop candidiasis in the esophagus, stomach, small intestine, and cecum after oral inoculation, but are resistant to systemic candidiasis (29, 30, 31, 32). Thus, host defense systems mediated by cells other than B and T lymphocytes, such as macrophages, can protect from hematogenous dissemination of C. albicans.

In this study we demonstrated that in vivo IL-13 and PPAR-{gamma} ligand treatment reduces C. albicans colonization of the esophagus and GI tract of immunocompetent and immunocompromised RAG-2–/– mice. Both treatments modulate the effector functions of macrophages, because peritoneal macrophages from IL-13 and PPAR{gamma} ligand-treated immunodeficient or immunocompetent mice increased their phagocytic activity and their ability to produce ROIs. Our data are consistent with previous in vitro studies which demonstrated an essential role for IL-13 and PPAR{gamma} ligands in macrophage activation to eliminate C. albicans, by increasing their ability to engulf C. albicans and to secrete ROIs during C. albicans challenge (6). Furthermore, we showed that treatment of immunocompetent or immunocompromised RAG-2–/– mice with IL-13 or PPAR-{gamma} ligands was efficient to induce overexpression of MRs on peritoneal macrophages. This MR induction by IL-13 and PPAR{gamma} ligands in in vivo treatments is stronger on macrophages harvested from RAG-2–/– mice than from wild-type mice. This result strongly suggest that in RAG-2–/– mice the innate immune response is more sensitive to compensate the suppression of lymphocyte populations. This increase of MR induction could depend on a greater sensitivity of PPAR{gamma} due to an increase of PPAR{gamma} activity and/or an overexpression of PPAR{gamma} level in macrophages from RAG-2–/– mice in presence of IL-13 or rosiglitazone. Interestingly, the increase of C. albicans phagocytosis by macrophages harvested from RAG-2–/– mice treated by rosiglitazone or IL-13 is also higher compared with macrophages from immunocompetent mice. Because the MR is the main receptor involved in yeast phagocytosis and because the mechanism of defense against C. albicans is mainly dependent on the ability of macrophages to phagocytose the yeasts, the increase of treatment efficacy in RAG-2–/– mice compared with immunocompetent mice is hence correlated with the higher capacity of macrophages to phagocytose the yeasts through MR. The existence of a true correlation between the increase in macrophage MR expression and the enhancement of their effector functions by IL-13 or PPAR{gamma} ligands is in line with our previous work which showed that the increase in C. albicans phagocytosis and ROI production following treatment with IL-13 or PPAR{gamma} ligands in vitro was antagonized by a MR mRNA-specific antisense (6).

The overexpression of MR was also confirmed by confocal microscopy on CD68+ cells recruited in GI mucosa of mice infected with C. albicans and treated with IL-13 or rosiglitazone. We also found that C. albicans inoculation in GI tract triggered the recruitment in cecal mucosa of CD68+ cells slightly expressing the MR. These results are in line with previous studies showing that C. albicans secretes chemotactic factors which favor infiltration of CD68+ cells, such as macrophages and neutrophils (33). Moreover, the treatment with either rosiglitazone or IL-13 increased the number of CD68 cells in the cecal mucosa expressing the MR on their surface. As neutrophils do not express the MR (34), and PPAR{gamma} agonists reduce the degree of neutrophil infiltration under early acute inflammatory conditions in GI mucosa (35), these CD68+ cells could well be macrophages. These results indicate that IL-13 and rosiglitazone treatment favor the infiltration of macrophages into the cecal mucosa with a high proportion of type 2 macrophages (M2). Thus, the present study and our previous in vitro data strengthen the evidence for a relationship between the increase of MR expression in macrophages of GI mucosa, the activation of their effector functions, and the elimination of yeast in cecal mucosa after treatment of mice with both IL-13 and PPAR-{gamma} ligands. Thus, macrophages recruited after Candida infection and IL-13 or PPAR{gamma} ligand treatment, fight against fungal infection and hence contribute to eliminating C. albicans from the GI tract.

Recently, we showed that in vitro regulation of macrophage MR expression by IL-13 and rosiglitazone is dependent on the PPAR{gamma}-signaling pathway (6). PPAR{gamma} is a nuclear receptor initially linked to the regulation of adipogenesis in fat tissue (36). It was previously shown that PPAR{gamma} can regulate monocyte gene expression and differentiation (16), and that IL-4, and more recently IL-13, induce the expression of the type B scavenger receptor CD36 through PPAR{gamma} activation (16, 37, 38, 39), hence facilitating the phagocytosis of Plasmodium falciparum-parasitized erythrocytes (37) and to decreasing malaria-induced TNF-{alpha} secretion (40). In addition to CD36 overexpression, PPAR{gamma} plays a critical role in the regulation of cholesterol homeostasis by controlling the expression of a network of genes that mediate cholesterol efflux from cells and its transport in plasma. Then, PPAR{gamma} induces ABCA1 expression and cholesterol removal from macrophages through a transcriptional cascade mediated by the nuclear receptor liver X receptor-{alpha} (LXR-{alpha}), where ligand activation of PPAR{gamma} leads to primary induction of ABCA1 (41, 42). It is interesting to note that recent findings have established that genomic interrelationships between genes coding for PPAR{gamma} and LXR-{alpha} regulate the innate immune response against pathogens by exerting both positive and negative regulation of specific macrophage gene expression networks (42, 43, 44). Thus, LXR displays anti-inflammatory activities and promotes macrophage survival in settings of bacterial infection (45). Overall, these data support our hypothesis of PPAR{gamma} involvement in the elimination of GI candidiasis. In this study, the treatment of mice with GW9662, a specific and irreversible PPAR{gamma} antagonist, and the use of PPAR{gamma}+/– mice demonstrated the involvement of this nuclear receptor in the effects of IL-13 and rosiglitazone treatments on the increase of macrophage MR expression, ROI secretion, and phagocytosis. Indeed, we showed in PPAR{gamma}+/– mice that IL-13 and PPAR{gamma} ligand treatment induced only half of the increase of MR expression and reduced yeasts colonization of the GI tract less efficiently. As demonstrated by PCR and Western blot, PPAR{gamma}+/– mice express half the PPAR{gamma} mRNA and protein compared with homozygous PPAR{gamma}+/+ mice (46). This heterozygous model has already been used to study the physiological effects of PPAR{gamma} deficiency in mice, and, for example, to reveal the role of PPAR{gamma} in the therapeutic effects of 5-aminosalicylic acid as an anti-inflammatory drug used in bowel disease treatment (47). Finally, we demonstrated that pretreatment of animals with the specific PPAR{gamma} antagonist completely inhibited the protective effects of IL-13 and rosiglitazone against C. albicans GI colonization and abolished macrophage MR induction, indicating the involvement of PPAR{gamma} in these mechanisms. Other studies have shown that coadministration of GW9662 and rosiglitazone in vivo abolished the protective effect of rosiglitazone or 15d-PGJ2 on organ injury and dysfunction associated with septic shock (48, 49, 50). In the present study, we demonstrated that in the untreated control mice, the PPAR-{gamma}-specific antagonist (GW9662) did not modify macrophage MR expression which supports our previous in vitro works, showing that the GW9662 has no effect on basal expression of macrophage MR. This antagonist only inhibits the induction of macrophage MR expression by IL-13 and rosiglitazone (6). Thus, PPAR-{gamma} is only involved in up-regulation of macrophage MR and not in its basal expression. This point is particularly relevant to explain the role of PPAR-{gamma} in infection. These data clearly demonstrate that the role of PPAR-{gamma} in fungal elimination is dependent on macrophage MR overexpression. Interestingly, we obtained the same results with PPAR-{gamma}+/– mice. Indeed, PPAR-{gamma}+/– mice did not show any difference in susceptibility to C. albicans infection compared with PPAR-{gamma}+/+ mice. Likewise, the expression level of macrophage MR from PPAR-{gamma}+/– mice and their associated effector functions were not lower than in their wild-type littermates. PPAR-{gamma} therefore plays a role in MR expression and infectious processes only when it is activated by its ligand. Thus, PPAR-{gamma} is not involved in susceptibility to C. albicans infection but its activation promotes macrophage effector functions, and hence contributes to reducing yeast colonization.

In conclusion, the present results support our previous in vitro work showing that IL-13 positively regulates macrophage MR surface expression partly by controlling the production of PPAR{gamma} endogenous ligand, 15d-PGJ2, via a cPLA2 activation both in mouse (6) and in human monocytes (37). It shows the in vivo importance of the nuclear receptor PPAR{gamma} in the reduction of esophageal and GI candidiasis in immunocompetent and immunodeficient RAG-2–/– mice. Thus, the results suggest the possibility that PPAR{gamma} ligands may be of therapeutic value for esophageal and GI candidiasis in patients with compromised immune systems as they increase the innate immune response.


   Acknowledgments
 
We are grateful to A. Minty and Sanofi-Synthelabo Toulouse Labège for supplying IL-13. We thank Sophie Cassaing for helpful discussions and Valérie Bans, Philippe Batigne, Alain Jauneau, and Yves Martinez for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 A.C. was supported by SIDACTION, C.L. by the French Ministry of Education, Research and Technology. This work was also supported by a grant from the Tarn et Garonne Committee of the Ligue Contre le Cancer. Back

2 C.L. and A.C. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Bernard Pipy, Laboratoire Polarisation des Macrophages and Récepteurs Nucléaires dans les Pathologies Inflammatoires et Infectieuses, Université Paul Sabatier Toulouse III, INSERM IFR31, Institut Louis Bugnard, BP 84225, 31432 Toulouse cedex 4, France. E-mail address: Pipy{at}toulouse.inserm.fr Back

4 Abbreviations used in this paper: GI, gastrointestinal; ROI, reactive oxygen intermediate; MR, mannose receptor; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}; SDA, Sabouraud dextrose agar; CL, chemiluminescence; mBSA, mannosylated BSA; LXR-{alpha}, liver X receptor-{alpha}. Back

Received for publication April 12, 2007. Accepted for publication January 28, 2008.


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 Materials and Methods
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 Discussion
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